Method for producing endothelial cells

ABSTRACT

The present invention relates to a method for producing endothelial cells, including carrying out: (a) inducing a population of mesoderm-lineage cells containing endothelial progenitor cells from pluripotent stem cells without forming an embryoid body; and (b) culturing the population of mesoderm-lineage cells containing endothelial progenitor cells in the presence of RepSox, in this order. According to the present invention, endothelial cells with high quality can be efficiently produced from pluripotent stem cells. The endothelial cells obtained by the method of the present invention are useful for the production of, for example, a myocardial sheet, and expected to be utilized in the treatment of a heart disease. A myocardial sheet can be produced by mixing the endothelial cells obtained by the method of the present invention with myocardial cells and mural cells and culturing the cells.

TECHNICAL FIELD

The present invention relates to a method for producing endothelialcells with high quality from pluripotent stem cells.

BACKGROUND ART

A technical innovation associated with regenerative therapy, inparticular a method for preparing pluripotent stem cells, and a methodfor inducing differentiation of the pluripotent stem cells, have beendeveloped, so that studies targeting the regeneration of tissues oforganisms have been intensively carried out. Also as to the heart whichis indispensable for life maintenance of individual organisms, it isconsidered that a regenerative medical technology is utilized in thetreatment of heart diseases. For example, myocardial cells artificiallygenerated from pluripotent stem cells have been tried to be applied tothe treatment of heart diseases. At this time, the myocardial cells arenot used alone, but, for example, a method for forming a sheet-like cellconstruct containing myocardial cells has been developed (PatentPublication 1), and the usefulness of the myocardial sheet thus obtainedhas been shown in a model animal with myocardial infarction.

In Patent Publication 1, a myocardial sheet is produced by combiningmyocardial cells with endothelial cells, mural cells andFlk/KDR-positive cells and culturing the cells. Although the abovepublication discloses a method for preparing plural cells such asmyocardial cells, endothelial cells and mural cells as a cell mixture,it is desired to individually prepare these cells, from the viewpoint ofcontrol of the quality or the production process of a myocardial sheet.In addition, it has been expected that endothelial cells are used as amaterial for blood vessels formed in tissues when tissues or organsother than blood vessels are artificially constructed.

Several methods have been already known for the production ofendothelial cells. A method for efficiently producing endothelial cellsfrom pluripotent stem cells is, for example, a method described inPatent Publication 2. Although the method is a method of stepwiselyculturing pluripotent stem cells in plural media containing differentactive ingredients, there are still rooms for improvements in thequality of cells obtained.

PRIOR ART REFERENCES Patent Publications

Patent Publication 1: WO 2012/133945

Patent Publication 2: WO 2014/192925

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

In order to use endothelial cells as a material for producing amyocardial sheet as mentioned above and to use endothelial cellsthemselves as a medical composition or a material for a research, amethod for efficiently producing endothelial cells with higher qualityis desired. The term “endothelial cells with high quality” as usedherein refers to endothelial cells having one or more characteristicssuch as the cell numbers obtained are high, the purity is high (highpositive ratio of endothelial cell markers), the differentiation stageis homogenous, having excellent cell proliferation ability (for example,juvenile), being less susceptible to damage by freezing and thawing, andhaving small differences between lots.

The present invention intends to solve the problems owned by theconventional production methods, and an object of the present inventionis to provide a method for producing endothelial cells with highquality.

Means to Solve the Problems

As the result of intensive studies to solve the problems mentionedabove, the present inventors have found for the first time that when theendothelial cells are induced from pluripotent stem cells, a largeamount of high-purity endothelial cells can be obtained by culturingendothelial progenitor cells in the presence of a medium withoutcontaining RepSox. Further, the present inventors have revealed thatmost of the endothelial cells obtained by the above method are juvenileendothelial cells and keep high viability and proliferation rate evenafter cryopreservation. The present invention has been completed on thebasis of the above findings.

Concretely, the present invention relates to:

[1] a method for producing endothelial cells, including carrying out:(a) inducing a population of mesoderm-lineage cells containingendothelial progenitor cells from pluripotent stem cells without formingan embryoid body; and(b) culturing the population of mesoderm-lineage cells containingendothelial progenitor cells in the presence of RepSox, in this order.[2] the method according to the above [1], wherein the pluripotent stemcell is an embryonic stem cell (ES cell) or an induced pluripotent stemcell (iPS cell);[3] the method according to the above [1] or [2], wherein in the step(a) the pluripotent stem cell is sequentially cultured in(i) a medium containing activin A,(ii) a medium containing bone morphogenetic protein 4, and(iii) a medium containing vascular endothelial growth factor;[4] the method according to the above [3], wherein (ii) the mediumcontaining bone morphogenetic protein 4 further contains basicfibroblast growth factor;[5] the method according to any one of the above [1] to [4],characterized by further including, subsequent to the step (a), (a′)isolating endothelial progenitor cells from the population ofmesoderm-lineage cells;[6] the method according to the above [5], wherein in the step (a′)kinase insert domain receptor-positive cells are isolated as endothelialprogenitor cells;[7] the method according to any one of the above [1] to [6], wherein,prior to the step (b), the population of mesoderm-lineage cellscontaining endothelial progenitor cells or the isolated endothelialprogenitor cells are cultured in a medium without RepSox;[8] the method according to any one of the above [1] to [7],characterized by further including, subsequent to the step (b), (c)freezing endothelial cells;[9] a method for producing a myocardial sheet, including:producing endothelial cells according to a method as defined in any oneof the above [1] to [8], andmixing the endothelial cells with myocardial cells and mural cells toculture the cells; and[10] a cell composition containing kinase insert domainreceptor-positive endothelial progenitor cells and RepSox.

Effects of the Invention

According to the method of the present invention, endothelial cells withhigh quality can be efficiently produced from pluripotent stem cells.The endothelial cells obtained according to the method of the presentinvention are useful in the production of, for example, a myocardialsheet, and expected to be used for the treatment of a heart disease.Further, the cells can be used as a cell model of diseases caused byabnormality or the like of endothelial cells, or as a material of safetyevaluation for drugs. In addition, a myocardial sheet can be produced bymixing the endothelial cells obtained by the method of the presentinvention with myocardial cells and mural cells, and culturing thecells.

MODES FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail hereinbelow.

The term “pluripotent stem cell” as used herein refers to a stem cellhaving pluripotency capable of differentiating into plural kinds ofcells, and also having self-proliferation ability, and the pluripotentstem cell includes, for example, but not limited to, an inducedpluripotent stem cell (iPS cell), an embryonic stem cell (ES cell), agermline stem cell (GS cell), an embryonic germ cell (EG cell), anembryonic stem cell derived from cloned embryo obtained by nucleartransfer (nuclear transfer ES cell; ntES cell), a fused stem cell, andthe like. A preferred pluripotent stem cell is an iPS cell or an EScell, and a more preferred pluripotent stem cell is an iPS cell.

The iPS cell is a somatic-cell-derived artificial stem cell, which hasalmost the same properties as an ES cell, for example, differentiationpluripotency and proliferation ability by self-replication, and the iPScell can be produced by introducing specific nuclear reprogrammingsubstances in the form of nucleic acids or proteins into a somatic cellor by increasing expression levels of endogenous mRNAs and/or proteinsof the nuclear reprogramming substances with agent(s) (K. Takahashi andS. Yamanaka (2006), Cell, 126: 663-676; K. Takahashi et al. (2007),Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; and M.Nakagawa et al. (2008), Nat. Biotechnol., 26: 101-106). The nuclearreprogramming substance may be a gene specifically expressed in an EScell, or a gene or a gene product thereof playing an important role inmaintenance of undifferentiated state of an ES cell. The substanceincludes, for example, but not particularly limited to, Oct3/4, Klf4,Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc,N-Myc, TERT, SV40 large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28,Lin28b, Nanog, Esrrb or Esrrg. These nuclear reprogramming substancesmay be used in combination when establishing iPS cell. For example, thecombination includes at least one, two or three, and preferably thecombination includes four of the above nuclear reprogramming substances.

Human iPS cell line established as a cell line may be used in theembodiment of the present invention. A preferred human iPS cell line is,but not particularly limited to, ChiPSC7, ChiPSC11, ChiPSC12, ChiPSCl9,ChiPSC20, ChiPSC21, ChiPSC22, ChiPSC23, 201B7, 201B7-Ff, 253G1, 253G4,1201C1, 1205D1,1210B2 and 836B3, and a more preferred human iPS cellline is ChiPSC12. The above mentioned human iPS cell lines are availablefrom Cellartis, or iPS Academia Japan, Inc., or Center for iPS CellResearch and Application, Kyoto University.

The ES cell is a stem cell which has been established from inner cellmass of early embryo (for example, blastocyst) of a mammal such as humanor mice, and has differentiation pluripotency and proliferation abilityby self-replication. The ES cell has been discovered in mice in 1981 (M.J. Evans and M. H. Kaufman (1981), Nature 292: 154-156), andsubsequently, the ES cell has been established also in primates such ashuman and monkey.

The ES cell can be established by extracting an inner cell mass from ablastocyst of a fertilized egg of a subject animal and culturing theinner cell mass on a feeder of fibroblasts. In addition, the maintenanceof cells by subculturing can be carried out by using a mediumsupplemented with a substance such as LIF or bFGF. The method forestablishment and maintenance of ES cell of human and monkey isdescribed in, for example, H. Suemori et al. (2006), Biochem. Biophys.Res. Commun., 345: 926-932; H. Kawasaki et al. (2002), Proc. Nati. Acad.Sci. USA, 99: 1580-1585, and the like. In addition, some researchorganizations distribute ES cells. For example, KhES-1, KhES-2 andKhES-3 which are human ES cell lines are available from Institute forFrontier Medical Science, Kyoto University (Kyoto, Japan).

The culturing of pluripotent stem cells is carried out by preferablysubjecting pluripotent stem cells prepared according to any methods toan adhesion culture in an appropriate culture vessel/culture substrate.Here, the adhesion culture refers to the culturing of cells in a statein which cells are adhered to a culture vessel/culture substrate. In theadhesion culture, an embryoid body (EB) is not formed. The adhesionculture is carried out by using a culture vessel/culture substratecoated with a substance to which cells can be adhered. The substance towhich cells can be adhered includes, for example, various extracellularmatrices (collagen, gelatin, laminin, fibronectin, vitronectin,entactin, heparan sulfate proteoglycan, and the like), an alteredproduct or modified product thereof, polylysine, and a combinationthereof. Preferably, Matrigel(registered trademark) orSynthemax(registered trademark), each of which is a compositioncontaining plural extracellular matrices, is used in the presentinvention.

As to the culture vessel/culture substrate used in this step and otherculturing steps in the present invention, those having any kinds ofmaterials or shapes can be used so long as they do not inhibitmaintenance, viability, differentiation, maturation or self-replicationof the cells. The shape of the culture vessels/culture substrates isalso not limited, and those having any shapes such as a flask, a plate,a dish, a bag and an incubator can be used. Various commerciallyavailable culture vessels/culture substrates may be used. Further, theculturing utilizing a culture vessel provided with a hollow thread, or aculture substrate such as beads, may be carried out.

As a basal medium, a medium usable in the culturing of animal cells canbe used. For example, RPMI 1640 medium, DEF-CS medium, Medium 199,MCDB131 medium, IMDM, EMEM, aMEM, DMEM, Ham's F12 medium, Fischer'smedium, mixed media thereof or the like are used as a basal medium.Further, a Xeno-free medium or a synthetic medium (Chemically-Definedmedium) is also used. A commercially available medium sold as a mediumfor culturing pluripotent stem cells, for example, StemFit(registeredtrademark), mTeSR1, Essential8(trademark) or the like may be used.Preferably, DEF-CS medium is used as a basal medium. A medium may beadded with serum, or may be serum-free. Optionally, for example,albumin, transferrin, growth factors, KSR, N2 supplement, B27supplement, fatty acids, insulin, collagen precursor, lipids, aminoacids, vitamins, 2-mercaptoethanol, 3′-thiolglycerol, trace elements,antibiotics, antioxidants, buffers, inorganic salts, or the like may beadded thereto.

Cells at a density of, for example, from 0.5 to 20×10⁴ cells/cm²,preferably from 1 to 10×10⁴ cells/cm² are seeded to a culture vesselcoated with a substance to which cells can be adhered, and the cells arecultured in a suitable medium. The culturing period includes, forexample, 12 hours or more, preferably 24 hours or more, and morepreferably three days or more, but it is to be expected that anappropriate culturing period is determined depending upon the cells. Inaddition, a medium exchange or subculturing may be appropriately carriedout during the culturing period.

A method for separating pluripotent stem cells that have been subjectedto adhesion culture and other cells in the present invention from aculture vessel includes a physical method, a method using a chelator, anenzymatic method using a separating solution having a protease activityand/or a collagenase activity, for example, TrypLE(trademark) Select,Accutase(registered trademark), Accumax(registered trademark), or thelike, and a combination thereof. Preferably, after dissociating coloniesof pluripotent stem cells by an enzymatic method, a method forphysically subjecting cells to fine dispersion is carried out.Generally, the pluripotent stem cells which have been cultured to 80%confluent against the culture vessel used are subjected to a separatingoperation.

(1) Method for Producing Endothelial Cells of the Present Invention

(a) Step of Inducing Population of Mesoderm-Lineage Cells ContainingEndothelial Progenitor Cells from Pluripotent Stem Cells Without FormingEmbryoid Body

This step is a step of inducing differentiation from pluripotent stemcells to a population of mesoderm-lineage cells containing endothelialprogenitor cells. A population of mesoderm-lineage cells containingendothelial progenitor cells are induced from an iPS cell or an ES cellin a preferred embodiment of the present invention, and from an iPS cellin a particularly preferred embodiment. In this step, the above cellpopulation can be induced without forming an embryoid body by preferablycarrying out contact culture.

The term “population of mesoderm-lineage cells” as used herein means acell population containing mesodermal cells themselves and/or cellsgenerated by differentiation from mesodermal cells. The cells generatedby differentiation from mesodermal cells include hemangioblasts,mesenchymal stem cells, hematopoietic stem cells, endothelial progenitorcells, cardiac progenitor cells, and the like.

The term “endothelial progenitor cells” as used herein means cells whosedifferentiation is directed to endothelial cells. The endothelialprogenitor cells can be confirmed by analyzing expression patterns oftranscriptional factors or cell-surface antigens. For example,expression patterns of transcriptional factors or cell-surface antigens,alone or in a combination, are measured, in which their expressionlevels are not detected or low even if detected before induction ofdifferentiation, and are remarkably increased after the induction ofdifferentiation. Markers effective for confirming endothelial progenitorcells include, for example, kinase insert domain receptor (KDR), FOXF1,BMP4, MOX1, SDF1 and the like. Preferably, the endothelial cellprogenitor cells are cells expressing KDR. KDR is a molecule which isalso referred to as vascular endothelial growth factor receptor-2:VEGFR-2 or Flk-1 as another name, and plays an important role in aprocess of vascularization.

This step (the step of inducing a population of mesoderm-lineage cellscontaining endothelial progenitor cells from pluripotent stem cellswithout forming an embryoid body) is not particularly limited, and anappropriate method may be selected from known methods. A preferredmethod for the present invention includes a method including culturingpluripotent stem cells in the presence of activin A and/or bonemorphogenetic protein 4 (BMP4), to provide mesodermal cells. Morepreferably, a method to induce mesodermal cells from pluripotent stemcells by sequentially carrying out three-step culturing of “(i)culturing in a medium containing activin A,” “(ii) culturing in a mediumcontaining BMP4,” and “(iii) culturing in a medium containing vascularendothelial growth factor (VEGF)” is exemplified. This three-stepculturing will be hereinafter explained in detail.

(i) Culturing in Medium Containing Activin A

As one embodiment of the present invention, mesodermal cells can beefficiently induced by culturing pluripotent stem cells in a sandwichmethod with an extracellular matrix, prior to culturing the cells in amedium containing activin A. Matrigel can be used as an extracellularmatrix. Concretely, pluripotent stem cells are seeded at a density of,for example, from 1 to 20×10⁴ cells/cm², and preferably from 2 to 15×10⁴cells/cm² to a culture vessel coated with Matrigel, and the cells aresubjected to adhesion culture in, for example, DEF-CS medium for 2 to 3days. Thereafter, the medium is exchanged with a medium added withMatrigel which is diluted to, for example, from 1:10 to 1:300,preferably from 1:20 to 1:150, and more preferably from 1:40 to 1:80,and the cells are further cultured for 16 to 24 hours in which theentire pluripotent stem cells are coated with Matrigel. The medium ofthe Matrigel-coated pluripotent stem cells thus obtained is exchangedwith a medium containing activin A.

The concentration of activin A added to a medium is, for example, from10 to 1,000 ng/mL, preferably from 25 to 500 ng/mL, and more preferably50 to 200 ng/mL. In addition, other growth factors and the like may beadded to the medium within the range so as not to impair the effects ofthe present invention, and this addition is carried out preferably inthe absence of BMP4 and VEGF.

As a basal medium, a medium usable in the culturing of animal cells canbe used. For example, RPMI 1640 medium, DEF-CS medium, Medium199,MCDB131 medium, IMDM, EMEM, aMEM, DMEM, Ham's F12 medium, Fischer'smedium, mixed media thereof and the like are used as a basal medium. Acommercially available medium sold as a medium for culturing pluripotentstem cells may be used. Preferably, RPMI 1640 medium is used as a basalmedium. A medium may be added with serum, or may be serum-free.Optionally, for example, albumin, transferrin, growth factors, KSR, N2supplement, B27 supplement, fatty acids, insulin, collagen precursor,lipids, amino acids, vitamins, 2-mercaptoethanol, 3′-thiolglycerol,trace elements, antibiotics, antioxidants, buffers, inorganic salts orthe like may be added thereto.

A preferred medium includes RPMI 1640 medium added with L-glutamine, B27supplement and activin A. Here, L-alanyl L-glutamine dipeptide orGlutaMAX(trademark) can be added in place of L-glutamine added to theabove medium or another medium in the present invention. TheGlutaMAX(trademark) contains L-alanyl L-glutamine dipeptide. SinceL-alanyl L-glutamine dipeptide having a stable structure does notdecompose in the same manner as L-glutamine during the storage orculturing to form toxic ammonia, the L-alanyl L-glutamine dipeptide issuitable for cell culturing.

The pluripotent stem cells are subjected to adhesion culture in themedium mentioned above. The culturing temperature is, for example, butnot limited to, from 30° to 40° C., and preferably 37° C. The culturingis carried out under an atmosphere of a CO₂-containing air, and the CO₂concentration is preferably from 2 to 8%. The culturing time is, forexample, from 6 hours to 5 days, and preferably from 12 to 48 hours.

(ii) Culturing in Medium Containing BMP4

The pluripotent stem cells which have been cultured in a mediumcontaining activin A are subsequently subjected to culturing in a mediumcontaining BMP4. This culturing may be carried out in the same manner asthe culturing of the above (i), or may be carried out in a differentmanner from the above culturing. Preferably, the adhesion culture withan extracellular matrix, for example, the adhesion culture usingMatrigel is carried out. In a case where the culturing is carried out bythe same method as the above culturing, a medium may be exchanged, andthe culturing may then be continued in the same vessel.

The concentration of BMP4 added to the medium is, for example, from 0.5to 200 ng/mL, preferably from 1 to 100 ng/mL, and more preferably from 2to 50 ng/mL.

In a preferred embodiment of the present invention, basic fibroblastgrowth factor (bFGF) is further added to the medium. The concentrationof bFGF added to the medium usable in this embodiment is, for example,from 0.5 to 200 ng/mL, preferably from 1 to 100 ng/mL, and morepreferably from 2 to 50 ng/mL. Further, other growth factors and thelike may be added to the medium within the range so as not to impair theeffects exhibited by the present invention, and this addition is carriedout preferably in the absence of activin A and VEGF.

The medium used in this culturing can be prepared by appropriatelycombining the same basal medium as those mentioned above and variouscomponents. A preferred medium includes RPMI 1640 medium added withL-glutamine, B27 supplement, bFGF and BMP4.

The culturing temperature is, for example, but not limited to, from 30°to 40° C., and preferably 37° C. The culturing is carried out under anatmosphere of a CO₂-containing air, and the CO₂ concentration ispreferably from 2 to 8%. The culturing time is, for example, from 12hours to 5 days, and preferably from 2 to 4 days.

(iii) Culturing in Medium Containing VEGF

Subsequent to the culturing of the above (ii), cells are subjected toculturing in a medium containing VEGF. This culturing may also becarried out in the same manner as the culturing of the above (i) and(ii). Preferably, the adhesion culture with an extracellular matrix iscarried out.

The concentration of VEGF in the medium usable in this culturing is, forexample, from 10 to 2,000 ng/mL, and preferably from 50 to 500 ng/mL.Further, other growth factors and the like may be added to the mediumwithin the range so as not to impair the effects exhibited by thepresent invention, and this culturing is carried out preferably in theabsence of activin A and BMP4.

The medium used in this culturing can also be prepared by appropriatelycombining the same basal medium as those mentioned above and variouscomponents. A preferred medium includes RPMI 1640 medium added withL-glutamine, B27 supplement and VEGF.

The culturing temperature is, for example, but not limited to, from 30°to 40° C., and preferably 37° C. The culturing is carried out under anatmosphere of a CO₂-containing air, and the CO₂ concentration ispreferably from 2 to 8%. The culturing time is, for example, from 6hours to 5 days, and preferably from 12 to 48 hours.

By this culturing, the population of mesoderm-lineage cells containingendothelial progenitor cells are obtained. Here, the ratio ofendothelial progenitor cells (KDR positive ratio) is, for example, 40%or more, preferably 50% or more, and more preferably 60% or more oftotal cell numbers.

(a′) Step of Isolating Endothelial Progenitor Cells from Population ofMesoderm-Lineage Cells

This step is a step of isolating endothelial progenitor cells from apopulation of mesoderm-lineage cells containing endothelial progenitorcells obtained in the above-mentioned step. Here, it is also possiblenot to carry out this step when a ratio of endothelial progenitor cells(KDR positive ratio) is already sufficiently high in the population ofmesoderm-lineage cells containing endothelial progenitor cells obtainedin the above-mentioned step. It is preferable that this step (a′) iscarried out subsequent to the step (a).

The isolation of the endothelial progenitor cells in this step iscarried out by selectively capturing cells expressing endothelialprogenitor cell markers mentioned above with a fluorescence-activatedcell sorting (FACS), a magnetic-activated cell sorting (MACS), or thelike, and then collecting the captured cells. In the above-mentionedseparating means, it is advantageous that an antibody specificallybinding to endothelial progenitor cell markers is utilized. Therefore,for example, an antibody or a fragment thereof that binds to KDR orother markers expressed on a surface of endothelial progenitor cells isutilized in the present invention.

Concretely, a cell population containing endothelial progenitor cellsare contacted with an appropriately labeled antibody which specificallybinds to an endothelial progenitor cell marker, for example, an anti-KDRantibody. Thereafter, the isolation of endothelial progenitor cells isachieved by collecting the labeled cells. For example, antibody-boundcells can be isolated by subjecting a cell population contacted with anfluorescence labeled antibody to a separation by FACS. In addition, in acase where an antibody labeled with a magnetic material is used, MACS orthe like can be utilized. Further, the above antibody may be combinedwith another antibody (secondary antibody) specifically binding to thelabel attached to the above antibody or the antibody itself. In thiscase, the separation of cells is carried out by using a label attachedto the secondary antibody. A commercially available antibody can be usedas the above anti-KDR antibody or the secondary antibody. For example,“anti-KDR antibody bonded with a PE fluorescent label” and “magneticbeads embedding anti-PE antibodies” can be used, without beingparticularly limited thereto.

The ratio of the isolated endothelial progenitor cells (KDR positiveratio) is, for example, 70% or more, preferably 80% or more, and morepreferably 90% or more.

The isolated endothelial progenitor cells can be directly differentiatedto endothelial cells, or the endothelial progenitor cells can bedifferentiated to endothelial cells after maintenance culturing onetime, while keeping the differentiation state of the endothelialprogenitor cells.

In a case where the endothelial progenitor cells are subjected tomaintenance culturing, while keeping the differentiation state of theendothelial progenitor cells, the maintenance culturing may be carriedout in the same manner as the culturing in the above-mentioned step.Concretely, the maintenance culturing is carried out by the adhesionculture using an extracellular matrix such as Matrigel. The medium usedin this maintenance culturing can also be prepared by appropriatelycombining the same basal medium as those mentioned above with variouscomponents. A commercially available medium sold as a medium forculturing endothelial cells, for example, a medium for proliferatingendothelial cells or the like may be used. A preferred medium includesRPMI 1640 medium added with L-glutamine, B27 supplement and VEGF. Themedium may further contain antibiotic such as penicillin orstreptomycin, or ROCK inhibitor. The ROCK inhibitor is not particularlylimited so long as the inhibitor can inhibit the function of Rho kinase(ROCK), and the inhibitor includes, for example, Y-27632. Theconcentration of Y-27632 is, for example, from 1 to 500 μM, preferablyfrom 3 to 200 μM, and more preferably from 10 to 50 μM. Further, othergrowth factors and the like may be added to the medium within the rangeso as not to impair the effects exhibited by the present invention, andthis maintenance culturing is carried out preferably in the absence ofRepSox mentioned below in detail.

The culturing time of the maintenance culturing includes, for example,24 hours or more, preferably 2 days or more, and more preferably 3 daysor more, and it is to be expected that an appropriate culturing time isdetermined depending upon the state of cells or culturing conditions. Inaddition, a medium exchange or subculturing may be appropriately carriedout during the culturing period.

Usually, the number of endothelial progenitor cells does not show aremarkable increase or decrease in this step of maintenance culturing.In addition, the ratio of endothelial progenitor cells (KDR positiveratio) is hardly decreased by the maintenance culturing. For example,after the maintenance culturing is carried out for three days, the ratioof the endothelial progenitor cells (KDR positive ratio) is 70% or more,preferably 80% or more, and more preferably 90% or more.

(b) Step of Culturing Population of Mesoderm-Lineage Cells ContainingEndothelial Progenitor Cells in the Presence of RepSox

This step is a step of culturing the population of mesoderm-lineagecells containing endothelial progenitor cells obtained in the stepmentioned above in the presence of RepSox. In a case where the step (a′)is carried out, the endothelial progenitor cells are cultured in thepresence of RepSox in this step.

The population of mesoderm-lineage cells containing endothelialprogenitor cells or endothelial progenitor cells obtained in the stepmentioned above are subsequently subjected to culturing in a mediumcontaining RepSox. This culturing step may be carried out in the samemanner as the culturing in the step mentioned above, or may be carriedout in a different manner from the culturing in the step mentionedabove. Preferably, the adhesion culture with an extracellular matrix iscarried out. In a case where the culturing is carried out in the samemanner as the culturing in the step mentioned above, a medium isexchanged, and the culturing may then be continued in the same vessel.In a case where a culture vessel coated with Matrigel is used, forexample, a population of mesoderm-lineage cells containing endothelialprogenitor cells or endothelial progenitor cells are seeded at a densityof from 0.5 to 10×10⁴ cell/cm², and preferably from 1 to 5×10⁴ cell/cm²,and the cells are cultured in a medium containing RepSox. Sinceendothelial progenitor cells are differentiated into endothelial cellsfor the first time by using RepSox in this step, it is preferable thatthe population of mesoderm-lineage cells containing endothelialprogenitor cells or isolated endothelial progenitor cells are culturedin a medium without containing RepSox, prior to this step (b).

RepSox (CAS No. 446859-33-2) is a low-molecular weight compound which isalso referred to as E-616452, SJN2511, Alk5 Inhibitor II, TGF-βRI KinaseInhibitor II, or the like as another name. RepSox is a strong selectiveinhibitor of ALK5, one of TGF-β receptors, which suppresses the functionof TGF-β by inhibiting ALK5. RepSox is one of TGF-β inhibitors. Here,the transforming growth factor (TGF)-β is one of naturally occurringgrowth factors having many characteristics, that plays in an importantrole in tissue development, cell differentiation, embryonic growth, andthe like.

SB431542 (CAS No. 301836-41-9) is a low-molecular weight compound thatinhibits ALK4, ALK5 and ALK7, each of which is TGF-β receptor. SB431542suppresses the function of TGF-β by inhibiting a group of receptorsmentioned above. SB431542 is one of TGF-β inhibitors.

The concentration of RepSox usable in this step is not particularlylimited so long as RepSox can achieve the desired effects such asinducing endothelial cells in high efficiency. The concentration ofRepSox added to a medium used in this step is, for example, from 0.5 to100 μM, preferably from 1 to 50 μM, and more preferably from 3 to 30 μM.

The medium used in this step can also be prepared by appropriatelycombining the same basal medium as those mentioned above with variouscomponents. A preferred medium includes a medium for proliferating forendothelial cells added with RepSox.

The culturing time in the presence of RepSox includes, for example, from24 hours to 14 days, and preferably from 2 to 7 days, and it is to beexpected that an appropriate culturing time is determined depending uponthe concentration of RepSox. In addition the concentration of RepSox maybe appropriately changed.

The population of mesoderm-lineage cells containing endothelialprogenitor cells or endothelial progenitor cells are cultured in thepresence of RepSox, whereby the proliferation of endothelial progenitorcells can be promoted, and as a result, endothelial cells expressingendothelial cell markers can be obtained in a large amount. In addition,the endothelial cells obtained in this step have one or morecharacteristics such as the cell numbers obtained are high, the purityis high (high positive ratio of endothelial cell markers), thedifferentiation stage is homologous, having excellent cell proliferationability (for example, being juvenile), being less susceptible to damageby freezing and thawing, and having small differences between lots.

The term “endothelial cells” as used herein means cells expressing atleast one of endothelial cell markers such as PE-CAM (CD31), VE-cadherin(CD144), Endoglin (CD105) and von Willebrand factor (vWF). Here, cellsexpressing CD31 are preferred, and cells expressing both of CD31 andCD144 are more preferred. The endothelial cells obtained by the presentinvention contain CD31-positive cells in a ratio of, for example, 70% ormore, preferably 80% or more, and more preferably 90% or more, withoutparticularly limiting the present invention thereto.

The endothelial cells can be more finely classified depending upondifferentiation stages thereof. Among the endothelial cells, cells thatare more undifferentiated are called as “juvenile endothelial cells,”and cells with progressed differentiation are called as “matureendothelial cells.” The differentiation stage of endothelial cells canbe confirmed by analyzing expression patterns of transcriptional factorsor cell-surface antigens. Concretely, the expression patterns oftranscriptional factors or cell-surface antigens, alone or in acombination, are measured, in which their expression levels areremarkably changed depending upon the progress of differentiation ofendothelial cells. Markers effective for confirming the differentiationstage of endothelial cells include, for example, the above-mentionedendothelial cell markers (CD31, CD144, CD105, vWF) and CD34. CD34 is amarker of hematopoietic stem cells or endothelial progenitor cells. Inaddition, CD34 is also expressed on the juvenile endothelial cells. Thecells expressing both of CD31 and CD34 (hereinafter, CD31+/CD34+ cells)are, but not particularly limited to, one example of juvenileendothelial cells. The endothelial cells obtained by the presentinvention contain juvenile endothelial cells in a large amount, and, forexample, the cells contain CD31+/CD34+ cells in a ratio of 70% or more,preferably 80% or more, and more preferably 90% or more, withoutparticularly limiting the present invention thereto.

According to the method for producing endothelial cells of the presentinvention, various endothelial cells such as vascular endothelial cells,lymphatic endothelial cells or corneal endothelial cells can beproduced.

Larger amounts of endothelial cells can be obtained by continuing theculturing of endothelial cells. As a medium used in this step, a mediumprepared by appropriately combining a basal medium and variouscomponents may be used, and it is preferable that this step is carriedout in the absence of RepSox. A commercially available medium sold as amedium for culturing endothelial cells, for example, a medium forproliferating endothelial cells may be used. For example, endothelialcells can be produced by using a medium for proliferating endothelialcells, and continuing the culturing while carrying out medium exchangeand subculturing every 1 to 3 days. The endothelial cells thus producedcan maintain the properties of endothelial cells for a long term.

In addition, a large decrease of endothelial cell markers is notobserved in this step (b). The endothelial cells can maintain, but notparticularly limited to, a CD31 positive ratio exceeding 60%, andpreferably a CD31 positive ratio exceeding 80%, even 30 days after thebeginning of the induction of differentiation.

(c) Step of Freezing Endothelial Cells

This step is a step which is carried out after step (b), and is a stepof freezing endothelial cells obtained in a series of the stepsmentioned above. The freezing of endothelial cells is carried out bysuspending endothelial cells in a solution containing a cryoprotectant,dispensing this suspension in an appropriate storage vessel, andfreezing the storage vessel according to an appropriate method.

As a cryoprotectant usable in the step of freezing endothelial cells,any of cryoprotectants can be used so long as the cryoprotectants do notinhibit the maintenance, viability, differentiation, maturation orself-replication of the cells. For example, glycerol, ethylene glycol,dimethyl sulfoxide, sucrose, glucose, polyvinyl pyrrolidone, trehaloseor the like can be used. Various commercially available cryoprotectantsmay also be used. For example, STEM-CELLBANKER(registered trademark),manufactured by Nippon Zenyaku Kogyo Co., Ltd., and preferablyCELLBANKER(registered trademark), manufactured by Nippon Zenyaku KogyoCo., Ltd. are used as a cryoprotectant. For example, endothelial cellsare, but not particularly limited to, suspended in CELLBANKER so as tohave a concentration of from 0.5 to 10×10⁶/mL, and preferably from 1 to5×10⁶/mL, and the suspension is dispensed to a storage vessel.

As a storage vessel, those having any materials and shapes can be usedso long as the vessel does not inhibit the maintenance, viability,differentiation, maturation or self-replication of the cells. The shapeof the storage vessel also is not particularly limited, and the vesselhaving any shapes such as vial, flask or bag can be used. Variouscommercially available storage vessels may be used. For example, a vialis used as a storage vessel.

As a freezing method, any methods are carried out so long as the methoddoes not inhibit the maintenance, viability, differentiation, maturationor self-replication of the cells. For example, slow freezing is carriedout, without being particularly limited thereto. Slow freezing can alsobe carried out with a programmed freezer, and slow freezing can also becarried out by using a frozen-treated vessel such as BICELL.

The frozen endothelial cells may be stored in a liquid nitrogen, a deepfreezer held at −80° C., or the like. When the cells are stored in aliquid nitrogen, the endothelial cells can be stored for, for example,24 hours or more, preferably 3 days or more, and more preferably 2 weeksor more.

As a method for thawing the frozen endothelial cells, any of methods arecarried out so long as the methods do not inhibit the maintenance,viability, differentiation, maturation or self-replication of cells. Forexample, the cells are warmed with a water bath warmed to 37° C. andthawed, without being particularly limited thereto. The viability of thethawed endothelial cells is 60% or more, preferably 70% or more, andmore preferably 80% or more.

The culturing of the thawed endothelial cells may be carried out in thesame manner as the culturing of other cells in the present invention.Concretely, the culturing is preferably carried out by the adhesionculture with an extracellular matrix. A medium used in this culturingcan also be prepared by appropriately combining the same basal medium asthose mentioned above and various components. A preferred mediumincludes a medium for proliferating endothelial cells. The culturingtime includes, for example, 24 hours or more, preferably 3 days or more,and more preferably 7 days or more, and it is to be expected that anappropriate culturing time is determined depending upon an amount ofcells required. In addition, a medium exchange or subculturing may beappropriately carried out during the culturing period. For example, in acase where the culturing is carried out for 7 days, the endothelialprogenitor cells are increased to, for example, 1.1 times or more,preferably 1.3 times or more, and more preferably 1.5 times or more. Inaddition, a ratio of endothelial cells (CD31 positive ratio) is hardlydecreased. For example, after the culturing is carried out for 7 days,the ratio of endothelial cells (CD31 positive ratio) is 80% or more,preferably 90% or more, and more preferably 95% or more.

(2) Method for Producing Myocardial Sheet of the Present Invention

The endothelial cells obtained by the present invention mentioned abovecan be used for production of a myocardial sheet containing endothelialcells as a constituent. For example, Japanese Patent Laid-Open No.2012-210156 describes a method for producing a myocardial sheet in whichFlk/KDR-positive cells are mixed with myocardial cells, endothelialcells and mural cells to form a sheet. A myocardial sheet can beproduced in accordance with a method described in Japanese PatentLaid-Open No. 2012-210156 using the endothelial cells produced in thepresent invention, without particularly limiting the present inventionthereto.

The term “myocardial sheet” as used herein refers to a sheet-like cellconstruct composed of various cells forming the heart or blood vessels,in which cells are connected with each other via intercellular binding.Here, various cells forming the heart or blood vessels includemyocardial cells, endothelial cells and mural cells. A method forproducing a myocardial sheet in accordance with the method described inJapanese Patent Laid-Open No. 2012-210156 will be hereinafter explained.

<Step of Producing Myocardial Cells>

This step is the step of producing myocardial cells from pluripotentstem cells, which is achieved by a known method. Myocardial cells can beobtained by, for example, culturing pluripotent stem cells in accordancewith a method described in WO 2012/133954, without being particularlylimited thereto.

<Step of Producing FLK-positive Cells and Mural Cells>

FLK-positive cells can be obtained by culturing pluripotent stem cellsin accordance with any methods, for example, a method described inYamashita et al. (2000), Nature, 408: 92-96. In addition, a mixture ofcells of endothelial cells and mural cells can be obtained by culturingFLK-positive cells on the basis of the description of the samereference. Further, mural cells can be isolated by using an antibodyspecifically binding to a mural cell marker. Here, the mural cells meancells exhibiting properties equivalent to those of blood vessel wallcells (cells surrounding vascular endothelial cells from outsidethereof) or progenitor cells thereof.

<Step of Producing Myocardial Sheet>

FLK-positive cells are cultured on a temperature-sensitive culturevessel, for example UpCell, manufactured by CellSeed Inc., or the like.After the cells are cultured for 1 to 7 days, for example, after 3 days,the mixture of cells of endothelial cells and mural cells prepared bythe method of the present invention, and myocardial cells are added to aculture vessel in proper amounts. This mixture of cells is cultured in amedium containing vascular endothelial growth factor (VEGF) for 1 to 10days, for example, 4 days, thereby forming a sheet-like cell construct.A myocardial sheet thus formed can be removed from the culture vessel byallowing a culture vessel to stand at room temperature, whereby themyocardial sheet can be collected. Further, the produced myocardialsheet is laminated, as desired.

The myocardial sheet thus produced can be used for the treatment of aheart disease, for example, heart failure, myocardial infarction,ischemic heart disease, myocarditis, various cardiomyopathy, or otherapplications. For example, as to a heart disease such as myocardialinfarction, a myocardial sheet is placed so as to cover desired sites ofthe heart, thereby carrying out the treatment. A myocardial sheet istaken to a heart tissue, to promote the recovery of function of theheart.

According to the present invention, in addition to a method forproducing endothelial cells and a method for producing a myocardialsheet, endothelial cells produced by the method and a myocardial sheetproduced by the method are also provided.

(3) Cell Composition of the Present Invention

A cell composition of the present invention contains kinase insertdomain receptor (KDR)-positive endothelial progenitor cells and RepSox,and the composition is used in the production of endothelial cells invitro.

The KDR-positive endothelial progenitor cells contained in the cellcomposition of the present invention can be prepared by differentiatingpluripotent stem cells in accordance with a known method. For example,the endothelial progenitor cells can be obtained by using pluripotentstem cells such as ES cells or iPS cells as a material, and carrying outstep (a) among the above method for producing endothelial cells of thepresent invention, or carrying out the step (a) and the step (b). Theabove step (a′) may be further carried out as needed. Here, the contentof the KDR-positive endothelial progenitor cells contained in the cellcomposition is, for example, but not particularly limited to, 70% ormore, preferably 80% or more, and more preferably 90% or more, of thecells that are KDR-positive.

The concentration of RepSox contained in the above cell composition isnot particularly limited so long as the desired effects such as inducingendothelial cells in high efficiency can be achieved. The concentrationof RepSox is, for example, from 0.5 to 100 μM, preferably from 1 to 50μM, and more preferably from 3 to 30 μM.

The above-mentioned cell composition can contain a medium prepared byappropriately combing the same basal medium as those used in other stepsin the present invention and various components. A preferred mediumincludes a medium for proliferating endothelial cells added with RepSox.

The above-mentioned cell composition is cultured for, for example, from24 hours to 14 days, and preferably from 2 to 7 days, thereby inducingthe differentiation from endothelial progenitor cells into endothelialcells, whereby endothelial cells expressing endothelial cell markers canbe obtained. It is to be expected that an appropriate culturing time isdetermined depending upon the concentration of RepSox used.

EXAMPLES

The present invention will be described more particularly by thefollowing Examples, without intending to limit the scope of the presentinvention to these Examples.

Example 1 Differentiation from iPS Cells into Vascular Endothelial Cells

Human iPS cells (ChiPSC12 strain) (Cellartis) were cultured in DEF-CSmedium (Cellartis) in accordance with the instructions attached to themedium.

In order to efficiently induce differentiation of mesodermal cells, iPScells were coated with Matrigel (Matrigel Sandwich method) in accordancewith the following procedures. First, TrypLE(trademark) Select (Lifetechnologies) was added to iPS cells that had been subcultured, and thecells were incubated at 37° C. for 3 to 7 minutes. The detached cellswere collected as a single cell by pipetting, and the cell numbers werecounted. Next, the cells were seeded at a density of 6×10⁴ cells/cm² toa culture vessel coated with Matrigel(trademark) (Corning), and culturedin DEF-CS medium for 2 to 3 days. Thereafter, the medium was exchangedwith DEF-CS medium added with Matrigel diluted to 1:60, and the cellswere cultured for additional 16 to 24 hours, thereby coating an upperlayer of the cells with Matrigel.

The Matrigel-coated iPS cells obtained were differentiated intomesodermal cells in accordance with the following procedures. First, amedium of the Matrigel-coated iPS cells was exchanged with RPMI 1640medium (Gibco) added with 100 ng/mL activin A (R&D), 2 mMGlutaMAX(trademark) (Thermo Fisher Scientific), and B27 supplement(without containing insulin) (Gibco) (this time point being defined asthe beginning of the induction of differentiation=Day 0), and the cellswere cultured for 24 hours (Day 1). Next, the medium was exchanged withRPMI 1640 medium added with 10 ng/mL human bone morphogenetic protein 4(hBMP4) (R&D), 10 ng/mL human basic fibroblast growth factor (hbFGF)(Peprotech), 2 mM GlutaMAX, and B27 supplement, and the cells werecultured for 3 days (Day 4). Thereafter, the medium was exchanged withRPMI 1640 medium added with 100 ng/mL vascular endothelial growth factor(VEGF) (Peprotech), 2 mM GlutaMAX, and B27 supplement, and the cellswere cultured overnight (Day 5). Here, as a result of observation of thecultured cells with an optical microscope, the formation of an embryoidbody could not be confirmed through Day 0 to Day 5.

Kinase insert domain receptor (KDR)-positive cells were separated fromthe cultured cells at Day 5 with a magnetic-activated cell sorting[MACS(registered trademark), Miltenyi biotech], thereby isolatingvascular endothelial progenitor cells. In order to separate theKDR-positive cells, an anti-KDR antibody (Miltenyi biotech) to which aPE fluorescent label was bound was used as a primary antibody forlabeling cells, and cells labeled with the primary antibody werecollected with magnetic beads (Miltenyi biotech) embedding anti-PEantibodies. When the ratio of KDR-positive cells existing in cells whichwere separated by MACS was measured with a flow cytometry, theKDR-positive ratio was from 89 to 99%. The cells obtained were seeded ata density of 2.7×10⁴ cells/cm² to a culture vessel coated with Matrigel,and cultured in RPMI 1640 medium added with B27 supplement, 50 units/mLpenicillin, 50 μg/mL streptomycin, 2 mM GlutaMAX, 20 μM Y-27632, and 100ng/mL VEGF for 16 to 24 hours (Day 6). Further, the medium of Day 6 wasexchanged with RPMI 1640 medium added with B27 supplement, 50 units/mLpenicillin, 50 μg/mL streptomycin, 2 mM GlutaMAX, and 100 ng/mL VEGF,and the cells were cultured for 2 days (Day 8).

The cells were collected at Day 8, and suspended in a medium forproliferating endothelial cells (Promocell) added with RepSox at aconcentration listed in Table 1. Thereafter, the cells were seeded at adensity of 2×10⁴ cells/cm² to a culture vessel, and cultured for 3 days(Day 11). Here, SB431542, another TGF-β inhibitor, was used as anegative control.

The cells were collected at Day 11, and the cell numbers were counted.The cells were suspended in a medium without containing a TGF-βinhibitor, i.e. a medium for proliferating endothelial cells, and seededat a density of 2×10⁴ cells/cm² to the culture vessel. The medium wasexchanged every 1 to 3 days, and the cells were collected at Day 19, andthe cell numbers were counted. At the same time, a part thereof was usedto measure the CD31 positive cell ratio, an endothelial cell marker,with a flow cytometry. The remaining cells were suspended in a mediumfor proliferating endothelial cells (without containing a TGF-βinhibitor), and the cells were then seeded at a density of 1×10⁴cells/cm² to a culture vessel. The medium was exchanged every 1 to 3days, the cells were collected at Day 30, and the cell numbers werecounted. At the same time, the CD31-positive cell ratio was measuredwith a flow cytometry.

The cell numbers at Day 19 and Day 30 are shown in Table 1 when the cellnumbers at Day 8 are defined as 1. In addition, the CD31-positive cellratios at Day 19 and Day 30 are shown in Table 2.

TABLE 1 Cell Cell Cell TGF-β Inhibitor Numbers Numbers Numbers (Days 8to 11) Concentration (Day 8) (Day 19) (Day 30) None 0 μM 1 ND NDSB431542 10 μM 1 1.3 0.9 (Negative Control) 20 μM 1 2.3 5.1 RepSox 3 μM1 3.1 2.5 10 μM 1 3.5 9.1 20 μM 1 3.5 3.1

TABLE 2 TGF-β Inhibitor CD31-Positive CD31-Positive (Days 8 to 11)Concentration (Day 19) (Day 30) SB431542 10 μM 97.7% 89.4% (NegativeControl) 20 μM 98.7% 33.0% RepSox 3 μM 98.9% 87.0% 10 μM 99.7% 99.0% 20μM 99.8% 99.1%

It was shown that regardless of the kinds of the TGF-β inhibitors used,the CD31-positive ratios at Day 19 exceeded 97%, so that vascularendothelial cells having high purity were obtained by the method of thepresent invention. When SB431542 was used, the cell proliferation rateat Day 30 was 0.9 in 10 μM SB431542. In addition, while the cellproliferation rate at Day 30 was 5.1 in 20 μM SB431542, theCD31-positive ratio was 33.0%. On the other hand, when RepSox was usedas a TGF-β inhibitor, the cell proliferation rates at Day 30 were 2.5 ormore and CD31-positive ratios were 87% or more in both the mediacontaining 3 μM RepSox. These results show that RepSox can satisfy bothof cell proliferation rate and purity at high levels in the induction ofdifferentiation of vascular endothelial cells, as compared to those ofSB431542, and therefore, RepSox is more useful. Here, since a sufficientamount of cells was not obtained at Day 11 in a medium without adding aTGF-β inhibitor, the cells were not subcultured.

Example 2 Freezing and Thawing of Vascular Endothelial Cells

The induction of vascular endothelial cells was carried out inaccordance with a method using a medium added with 20 μM RepSoxdescribed in Example 1, and the cells were collected from Days 18 to 22.The collected cells were washed, and suspended in Stem Cell Banker(TAKARA BIO INC.) so as to have a cell concentration of 3×10⁶/mL, and 1mL of each of the suspension was dispensed to vials. The vials wereplaced in a frozen-treated vessel (BICELL, Nihon Freezer Co., Ltd.), andthe cells were subjected to slow freezing in a freezing storage chamberheld at a temperature of −80° C. Thereafter, each vial was transferredto a liquid nitrogen, and stored for 3 days.

The frozen cells were warmed for 2 minutes and 30 seconds with a waterbath warmed to 37° C., to thaw the cells. The cell viability wasmeasured by using a part of the cells, and as a result, the cellviability was 87%. An entire amount of the remaining cells was added toa tube to which 8 mL of a medium for proliferating endothelial cells hadbeen dispensed in advance. Thereafter, a vial was rinsed with 1 mL of amedium for proliferating endothelial cells, and a rinsed medium wasadded to the tube. The tube was centrifuged at 200×g for 5 minutes, andthe supernatant was then discarded. The cells were suspended in a freshmedium for culturing endothelial cells, and the cells were seeded at adensity of 2×10⁴ cells/cm² to a culture vessel. The medium was exchangedon the next day, and the medium was then exchanged every 1 to 3 days.

The cells were collected on a seventh day after thawing the cells, andthe cell numbers were counted. At the same time, the CD31-positive cellratio, an endothelial cell marker, was measured with a flow cytometry.As a result, the cell proliferation rates of from 1.5 to 4 times and theCD31 positive ratios of 95% or more (96.6 to 99%) were found. It wasclarified from the results that the endothelial cells obtained by themethod of the present invention had a very small damage by freezing andthawing.

Example 3 Measurement of CD31+/CD34+ Cells

Human iPS cells (ChiPSC12 strain) were cultured in DEF-CS medium inaccordance with the instructions attached to the medium.

TrypLE(trademark) Select was added to iPS cells that had beensubcultured, and the cells were incubated at 37° C. for 3 to 7 minutes.The detached cells were collected as a single cell by pipetting, and thecell numbers were counted. Next, the cells were seeded at a density of6×10⁴ cells/cm² to a culture vessel coated with Matrigel(trademark), andcultured in DEF-CS medium for 2 to 3 days. Thereafter, the medium wasexchanged with DEF-CS medium added with Matrigel diluted to 1:60, andthe cells were cultured for additional 16 to 24 hours, thereby coatingan upper layer of the cells with Matrigel.

The Matrigel-coated iPS cells obtained were differentiated intomesodermal cells in accordance with the following procedures. First, amedium of the Matrigel-coated iPS cells was exchanged with RPMI 1640medium added with 100 ng/mL activin A, 2 mM GlutaMAX(trademark), and B27supplement (without containing insulin) (this time point being definedas the beginning of the induction of differentiation=Day 0), and thecells were cultured for 24 hours (Day 1). Next, the medium was exchangedwith RPMI 1640 medium added with 10 ng/mL hBMP4, 10 ng/mL hbFGF, 2 mMGlutaMAX, and B27 supplement, and the cells were cultured for 3 days(Day 4). Thereafter, the medium was exchanged with RPMI 1640 mediumadded with 100 ng/mL VEGF, 2 mM GlutaMAX, and B27 supplement, and thecells were cultured overnight (Day 5). Here, as a result of observationof the cultured cells with an optical microscope, the formation of anembryoid body could not be confirmed through Day 0 to Day 5.

KDR-positive cells were separated from the cultured cells at Day 5 byusing the magnetic-activated cell sorting, thereby isolating vascularendothelial progenitor cells. In order to separate the KDR-positivecells, an anti-KDR antibody to which a PE fluorescent label was boundwas used as a primary antibody for labeling cells, and cells labeledwith the primary antibody were collected with magnetic beads embeddinganti-PE antibodies. When the ratio of KDR-positive cells existing in thecells separated by MACS was measured with a flow cytometry, the KDRpositive ratio was from 89 to 99%. The cells obtained were seeded at adensity of 2.7×10⁴ cells/cm² to a culture vessel coated with Matrigel,and cultured in RPMI 1640 medium added with B27 supplement, 50 units/mLpenicillin, 50 μg/mL streptomycin, 2 mM GlutaMAX, 20 μM Y-27632, and 100ng/mL VEGF for 16 to 24 hours (Day 6). Further, the medium at Day 6 wasexchanged with RPMI 1640 medium added with B27 supplement, 50 units/mLpenicillin, 50 μg/mL streptomycin, 2 mM GlutaMAX, and 100 ng/mL VEGF,and the cells were cultured for 2 days (Day 8).

The cells were collected at Day 8, and suspended in a medium forproliferating endothelial cells (Promocell) added with RepSox having aconcentration listed in Table 3. Thereafter, the cells were seeded at adensity of 2.7×10⁴ cells/cm² to a culture vessel, and cultured for 3days (Day 11).

The medium was exchanged with a medium without containing a TGF-βinhibitor, i.e. a medium for proliferating endothelial cells at Day 11,and the cells were collected at Day 13, and the cell numbers werecounted. At this time, a part of the cells was used to measureCD31-positive cells and CD34-positive cells with a flow cytometry. Theremaining cells were suspended in a medium for proliferating endothelialcells (without containing RepSox), and the cells were then seeded at adensity of 2.7×10⁴ cells/cm² to the culture vessel. The medium wasexchanged every 1 to 3 days, and the cells were collected at Day 13 andDay 18, and the cell numbers were counted. At the same time,CD31-positive cells and CD34-positive cells were measured with a flowcytometry.

Here, CD31 is a marker of endothelial cells, and CD34 is a marker ofhematopoietic stems cells or endothelial progenitor cells. In addition,CD34 is also expressed in juvenile endothelial cells. Accordingly,CD31-positive and CD34-positive cells (hereinafter, CD31+/CD34+) in thepresent invention are considered to be cells of which differentiationstage has not progressed among the vascular endothelial cells, in otherwords, juvenile vascular endothelial cells. The CD31+/CD34+ cell ratiosat Day 13 and Day 18 are shown in Table 3.

TABLE 3 TGF-β Inhibitor CD31+/CD34+ CD31+/CD34+ (Day 8 to 11)Concentration (Day 13) (Day 18) RepSox 3 μM 96.9% 93.3% 10 μM 96.1%94.4% 20 μM 95.8% 96.1%

Regardless of the concentrations of RepSox, the CD31+/CD34+ cells were95% or more at Day 13. In addition, at Day 18, as the concentration ofRepSox became higher, the proportion of CD31+/CD34+ cells became higher.These results show that juvenile endothelial cells having a potential todifferentiate and mature into various endothelial cells are obtained inhigh proportions by using RepSox, in the induction of thedifferentiation of the vascular endothelial cells.

INDUSTRIAL APPLICABILITY

According to the present invention, the endothelial cells with highquality are provided. The endothelial cells of the present invention areparticularly useful in the production of a myocardial sheet.

1-10. (canceled)
 11. A method for producing endothelial cells,comprising carrying out: (a) inducing a population of mesoderm-lineagecells comprising endothelial progenitor cells from human pluripotentstem cells without forming an embryoid body; (a′) isolating endothelialprogenitor cells from the population of mesoderm-lineage cells; and (b)culturing isolated endothelial progenitor cells in the presence ofRepSox, in this order, wherein in the step (a) the human pluripotentstem cell is sequentially cultured in (i) a medium comprising activin A,(ii) a medium comprising bone morphogenetic protein 4, and (iii) amedium comprising vascular endothelial growth factor.
 12. The methodaccording to claim 11, wherein the human pluripotent stem cell is ahuman embryonic stem cell (human ES cell) or a human induced pluripotentstem cell (human iPS cell).
 13. The method according to claim 11,wherein (ii) the medium comprising bone morphogenetic protein 4 furthercomprises basic fibroblast growth factor.
 14. The method according toclaim 11, wherein in the step (a′) a kinase insert domainreceptor-positive cells are isolated as endothelial progenitor cells.15. The method according to claim 11, wherein, prior to the step (b),the population of mesoderm-lineage cells comprising endothelialprogenitor cells or isolated endothelial progenitor cells are culturedin a medium without RepSox.
 16. The method according to claim 11,characterized by further comprising, subsequent to the step (b), (c)freezing endothelial cells.
 17. A method for producing a myocardialsheet, comprising: producing endothelial cells according to a method asdefined in claim 11, and mixing the endothelial cells with myocardialcells and mural cells to culture the cells.
 18. The method according toclaim 12, wherein (ii) the medium comprising bone morphogenetic protein4 further comprises basic fibroblast growth factor.
 19. The methodaccording to claim 12, wherein in the step (a′) a kinase insert domainreceptor-positive cells are isolated as endothelial progenitor cells.20. The method according to claim 13, wherein in the step (a′) a kinaseinsert domain receptor-positive cells are isolated as endothelialprogenitor cells.
 21. The method according to claim 12, wherein, priorto the step (b), the population of mesoderm-lineage cells comprisingendothelial progenitor cells or isolated endothelial progenitor cellsare cultured in a medium without RepSox.
 22. The method according toclaim 13, wherein, prior to the step (b), the population ofmesoderm-lineage cells comprising endothelial progenitor cells orisolated endothelial progenitor cells are cultured in a medium withoutRepSox.
 23. The method according to claim 14, wherein, prior to the step(b), the population of mesoderm-lineage cells comprising endothelialprogenitor cells or isolated endothelial progenitor cells are culturedin a medium without RepSox.
 24. The method according to claim 12,characterized by further comprising, subsequent to the step (b), (c)freezing endothelial cells.
 25. The method according to claim 13,characterized by further comprising, subsequent to the step (b), (c)freezing endothelial cells.
 26. The method according to claim 14,characterized by further comprising, subsequent to the step (b), (c)freezing endothelial cells.
 27. The method according to claim 15,characterized by further comprising, subsequent to the step (b), (c)freezing endothelial cells.
 28. A method for producing a myocardialsheet, comprising: producing endothelial cells according to a method asdefined in claim 12, and mixing the endothelial cells with myocardialcells and mural cells to culture the cells.
 29. A method for producing amyocardial sheet, comprising: producing endothelial cells according to amethod as defined in claim 13, and mixing the endothelial cells withmyocardial cells and mural cells to culture the cells.
 30. A method forproducing a myocardial sheet, comprising: producing endothelial cellsaccording to a method as defined in claim 14, and mixing the endothelialcells with myocardial cells and mural cells to culture the cells.